Project Summary_3Helix, Inc. In this Phase II SBIR proposal, 3Helix Inc. proposes to develop a new user-friendly imaging agent that can detect denatured collagen in histological tissue section and in vivo animal imaging. Collagen is the major structural component of nearly all human tissues and organs, providing a sturdy and bioactive framework for cell growth and tissue formation. Although degradation of collagen takes place during normal tissue development and maintenance, excessive levels of collagen degradation and denaturation is intricately involved in a wide variety of diseases including cancer, atherosclerosis, arthritis, osteoporosis, nephritis, and fibrosis. The denatured collagen molecule is a hallmark indicator of inflammation and tissue injury in these diseases. However, this indicator is rarely used in biomedical research, due to the lack of reagents or methods that can readily detect it. The founders of 3Helix, Inc., Drs. Yang Li and Michael Yu, discovered a synthetic Collagen Hybridizing Peptide (CHP), which can specifically bind to denatured collagen molecules with high affinity: the CHP peptide is able to form a stable hybridized triple helix structure only with the unraveled collagen strands but not with intact collagen molecules. The discovery of CHP, and its binding to denatured collagen, has been patented, published in multiple high impact journals (PNAS, Nat. Commun., ACS Nano). Because collagen degradation is associated with a wide range of human pathology and injury, CHP?s potential to detect and monitor disease have drawn overwhelming interests from academics and clinicians, as well as from industry and major reagent vendors. During the Phase I period, 3Helix developed CHP into a histology staining reagent for research use, and successfully commercialized it in 2017. The product has been sold to over 100 users from 11 countries including some of the leading experts from world-renowned institutes. Despite this success, current CHP requires heating as a pre-conditioning process which complicates histological study particularly for automated process. Such pre-conditioning is detrimental to comparative in vivo study. Recent study suggests that dimeric form of CHP is able to detect degraded collagens that a conventional monomeric CHP cannot detect due to improved affinity made possible by mutivalency. In the Phase II period of the SBIR grant, 3Helix plans to make significant improvement to the current peptide design in order to make CHP easier to use in vitro without heat treatment, but more importantly to develop a first-of- its-kind near-infrared fluorescence (NIRF) probe for in vivo animal imaging. The new design, which is based on solid preliminary study, involves the dimeric CHP structure comprised of non-self-trimerizing peptide sequence. In addition to developing the new probe and demonstrating its use in disease detection and monitoring for in vitro and in vivo use, 3Helix will also produce optimized protocols for i) in vitro assay of degraded collagen in tissue section as well as for ii) in vivo imaging of mouse disease model. The successful outcome of this project will open new avenues in basic research, drug development, and medical diagnosis for over 30 different types of human diseases, including cancer, fibrosis, cardiovascular disease, and arthritis.